Members of the ligand-induced nuclear receptor superfamily are medically important regulators of cellular activity. Hormones initiate a receptor activation process leading to receptor redistribution and binding to specific DNA recognition sites in the promoter regions of their target genes. Using microscopy and fluorescent protein chimeras of nuclear receptors, we pioneered studies establishing that ligand binding regulates the subcellular targeting of glucocorticoid (GR), vitamin D (VDR), and retinoid X receptors (RXR). These studies demonstrated that ligand binding induces formation of multiple nuclear foci of GFP-GR, GFP-VDR, YFP-RXR, and GFP-ER. Studies on a cell line harboring a large array of MMTV-LTR indicated that human GR accumulates along this array, indicating that foci represent GR interaction with DNA binding sites. In addition, mutational analysis demonstrated a correlation between hormone-dependent nuclear foci formation and DNA binding for both the GFP-VDR and the GFP-GR. This notion was further supported by our co-localization and fluorescence energy transfer experiments (FRET). After calcitriol treatment, GFP-VDR and RXR-BFP co-localized in the nuclear foci and FRET demonstrated formation of VDR/RXR dimers at these foci. Dimerization incompetent GFP-VDR and RXR-BFP failed to co-localize and failed to generate FRET signal at the foci. Because VDR and RXR bind to DNA as heterodimer, this finding suggests a correlation between focal receptor accumulation and DNA-binding. Furthermore, dynamic microscopy experiments such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) revealed that both VDR and RXR move rapidly within the nucleus. Our ongoing studies employing FRET between fluorescent derivatives of calcitriol and GFP-VDR explore the importance of interactions between the VDR and calcitriol at the nuclear level. We have continued to use advanced microscopy techniques to address the roles of receptor trafficking between the cytoplasm and the nucleus in the regulation of hormone actions. Our dynamic microscopy and cell permeabilization experiments demonstrated that YFP?RXR as well as both unliganded and liganded GFP-VDR shuttle rapidly between the cytoplasm and the nucleus. We postulated that redirection of VDR and RXR signaling with nuclear exclusion and retention results in the development of abnormal cellular functions. To test this hypothesis, we generated mutants of the known nuclear localization sequence (NLS) of VDR, identified a NLS of the RXR, and generated a mutant nlsRXR-BFP. Microscopy showed that these NLS mutants remained in the cytoplasm and luciferase reporter assays indicated that they fail to activate transcription of reporter genes. The export of unliganded GFP-VDR was sensitive to treatment with leptomycin B (LMB), an inhibitor of cargo protein binding to the Crm-1 export receptor. Mutations of phenylalanines to alanines in the calreticulin-binding site of VDR (calGFP-VDR) also inhibited VDR export. Pretreatment with LMB decreased hormone-induced transcriptional activity of VDR from 29-fold to 13-fold, and the calGFP-VDR was completely inactive. These data provided the first demonstration of the importance of export for transcriptional activity of a nuclear receptor. Furthermore, we demonstrated that dimerization with RXR has a profound effect on VDR nuclear import and export. Coexpression of RXR-BFP with GFP-VDR promoted nuclear accumulation of GFP-VDR by influencing both nuclear import and retention. RXR-BFP also promoted hormone-dependent nuclear accumulation of the nlsGFP-VDR, and rescued its transcriptional activity. Dimerization incompetent mutant of RXR-BFP failed to alter GFP-VDR and nlsGFP-VDR distribution and activities. Coexpression of nlsRXR-BFP with GFP-VDR caused cytoplasmic retention of unliganded GFP-VDR. Calcitriol addition induced partial nuclear accumulation of both GFP-VDR and nlsRXR-BFP. Coexpression of nlsRXR-BFP also decreased transcriptional activity of GFP-VDR. In addition to RXR effect on VDR import, dimerization with RXR also impacted VDR export. Unlike the VDR export, the export of YFP-RXR was insensitive to LMB. Expressed with RXR-BFP, GFP-VDR became LMB insensitive and the export rate decreased. In contrast, the expression of dimerization incompetent mutant of RXR-BFP had no effect on GFP-VDR export. This was the first demonstration of RXR effect on the export of a dimerization partner. Together these findings suggest that dimerization with RXR regulates VDR localization and this effect is important for VDR functions. Our studies have changed the paradigm of VDR and RXR localization within the cell and introduced a dynamic model for VDR activation.